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The DNA modification is dynamic during the conversion from fibroblasts to neurons [Methylation]

ABSTRACT: Expression level of 5mC during the conversion from fibroblasts to neurons

ORGANISM(S): Mus musculus

PROVIDER: GSE88868 | GEO | 2016/10/21

SECONDARY ACCESSION(S): PRJNA349015

REPOSITORIES: GEO

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The DNA modification is dynamic during the conversion from fibroblasts to neurons [Hydroxymethylation]

Project description:Expression level of 5mC during the conversion from fibroblasts to neurons

2016-10-21 | GSE88867 | GEO

Linking immortalization to direct conversion of human fibroblasts to neurons

Project description:Direct conversion from fibroblasts to neurons is a potential cell replacement therapy for neurological disorders, and a variety of combinations of transcription factors have been tried. We notice that the efficiency of conversion from aging fibroblasts was much lower than in early stage cells, which is consistent with the notion that cellular senescence impairs conversion of fibroblasts to neurons. Here, we found that the transient knockdown of the p16Ink4a/p19Arf locus was sufficient to convert human fibroblasts to neurons. Futhermore, expression of hTERT alone, another mechanism behind immortalization, also induced neuron conversion. Our results show that the acquisition of immortality is a crucial step for the conversion of human fibroblasts into induced neurons. Transient knockdown of p16/p19 or p53 expression or exogenous overexpression of hTERT can induce primary fibroblasts to immortality. In the following, treated cells were cultured in neuron-induction medium. We can observe the morphology change and detect the neuronal markers. Also, some of the induced neurons could generate action potentials and neurotransmitter-induced currents in optimal conditions.

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The DNA modification is dynamic during the conversion from fibroblasts to neurons

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Direct conversion of fibroblasts to neurons by a new mechanism – p53 regulation

Project description:Direct conversion from fibroblast to neuron has recently been successfully induced bypassing the pluripotent state. However, the conversion takes a few months with low percentages of success. Here we found that depletion of p53, which can converted fibroblasts into three major neural lineages: neurons, astrocytes and oligodendrocytes. Furthermore, our method provided a high efficiency of conversion in aging fibroblasts, where published methods failed. This finding may help developing a prototype for neuron replacement therapy, including foraging people vulnerable to neurological disorders. p53 has been shown to inhibit reprogramming of fibroblasts to iPS cells, by depletion of p53 in human fibroblasts, we study the function of p53 in induced neuron process. By induction of p53 knockdown fibroblasts with special neuron medium, we can get mature neurons directly. In the induction process, many neurogenic transcription factors were up-regulated, and we prove that p21 is not involved in this process.

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MicroRNAs and Ascl1 facilitate direct conversion of porcine fibroblasts into induced neurons

Project description:Direct neuronal conversion describes the process of generating induced neurons from somatic cells such as fibroblasts by overexpressing cell type-specific transcription factors. This was first achieved by expressing Brn2, Ascl1 and MytL1 in mouse fibroblasts, and was later achieved in human cells by inclusion of additional factors such as NeuroD1. Here, we present the first protocol for directly converting porcine fibroblasts into induced neurons. We used lentivirus-mediated delivery of previously identified neuronal-specifying transcription factors and microRNAs and evaluated morphology and neuronal marker expression after ten days of conversion. We found that Ascl1 and microRNAs, miR-9/9* and miR-124 together generated more neuronal cells than other conditions tested. The porcine induced neurons expressed common mature markers such as MAP2 and Synaptophysin after four weeks of conversion. Transcriptomic analysis revealed that fibroblast-specific signatures were silenced early in the conversion process, while the neuronal programs increased and matured during conversion. We generated a heterogenous population of glutamatergic and GABAergic neurons.

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Project description:Forced expression of pro-neural transcription factors was shown to mediate direct neuronal conversion of human fibroblasts. Since neurons are postmitotic, the conversion efficiency represents an important parameter. Here we present a minimalist approach combining two factor neuronal programming with small molecule-based inhibition of GSK3ß and SMAD signaling, which gives rise to functional neuron-like cells (iNs) of various neurotransmitter phenotypes with an overall yield of up to >200% and a final neuronal purity of up to >80%. Timcourse of reprogramming of fibroblasts towards an neuronal phenotype in two independent fibroblast lines

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